The present invention relates generally to screening and identification of new bioactive molecules. More specifically, the present invention relates to methods of using optical detection and capillary array-based techniques for screening samples or libraries and recovering bioactive molecules having a desired activity or nucleic acid sequences encoding bioactive molecules.
There has been a dramatic increase in the need for bioactive compounds with novel activities. This demand has arisen largely from changes in worldwide demographics coupled with the clear and increasing trend in the number of pathogenic organisms that are resistant to currently available antibiotics as well as the need for new industrial processes for synthesis of compounds. For example, while there has been a surge in demand for antibacterial drugs in emerging nations with young populations, countries with aging populations, such as the U.S., require a growing repertoire of drugs against cancer, diabetes, arthritis and other debilitating conditions. The death rate from infectious diseases has increased 58% between 1980 and 1992 and it has been estimated that the emergence of antibiotic resistant microbes has added in excess of $30 billion annually to the cost of health care in the U.S. alone. (Adams et al., Chemical and Engineering News, 1995; Amann et al., Microbiological Reviews, 59, 1995). As a response to this trend pharmaceutical companies have significantly increased their screening of microbial diversity for compounds with unique activities or specificities.
The majority of bioactive compounds currently in use are derived from soil microorganisms. Many microbes inhabiting soils and other complex ecological communities produce a variety of compounds that increase their ability to survive and proliferate. These compounds are generally thought to be nonessential for growth of the organism and are synthesized with the aid of genes involved in intermediary metabolism. Such secondary metabolites that influence the growth or survival of other organisms are known as xe2x80x9cbioactivexe2x80x9d compounds and serve as key components of the chemical defense arsenal of both micro- and macroorganisms. Humans have exploited these compounds for use as antibiotics, antiinfectives and other bioactive compounds with activity against a broad range of prokaryotic and eukaryotic pathogens (Barnes et al., Proc. Nat. Acad. Sci. USA., 91, 1994).
The approach currently used to screen microbes for new bioactive compounds has been largely unchanged since the inception of the field. New isolates of bacteria, particularly gram positive strains from soil environments, are collected and their metabolites tested for pharmacological activity.
There is still tremendous biodiversity that remains untapped as the source of lead compounds. However, the currently available methods for screening and producing lead compounds cannot be applied efficiently to these under-explored resources. For instance, it is estimated that at least 99% of marine bacteria species do not survive on laboratory media, and commercially available fermentation equipment is not optimal for use in the conditions under which these species will grow, hence these organisms are difficult or impossible to culture for screening or re-supply. Recollection, growth, strain improvement, media improvement and scale-up production of the drug-producing organisms often pose problems for synthesis and development of lead compounds. Furthermore, the need for the interaction of specific organisms to synthesize some compounds makes their use in discovery extremely difficult. New methods to harness the genetic resources and chemical diversity of these untapped sources of compounds for use in drug discovery are very valuable.
A central core of modem biology is that genetic information resides in a nucleic acid genome, and that the information embodied in such a genome (i.e., the genotype) directs cell function. This occurs through the expression of various genes in the genome of an organism and regulation of the expression of such genes. The expression of genes in a cell or organism defines the cell or organism""s physical characteristics (i.e., its phenotype). This is accomplished through the translation of genes into proteins. Determining the biological activity of a protein obtained from an environmental sample can provide valuable information about the role of proteins in the environments. In addition, such information can help in the development of biologics, diagnostics, therapeutics, and compositions for industrial applications.
Accordingly, the present invention provides methods to access this untapped biodiversity and to rapidly screen for sequences and activities of interest utilizing recombinant DNA technology. This invention combines the benefits associated with the ability to rapidly screen natural compounds with the flexibility and reproducibility afforded with working with the genetic material of organisms.
The invention provides a rapid and efficient method for identifying a bioactivity or biomolecule of interest. In one embodiment, the method includes introducing a recombinant clone into a capillary tube of a capillary array, wherein each capillary tube of the capillary array has at least one wall defining a lumen for retaining the recombinant clone, and wherein the at least one wall is made of a material having a low refractive index. The recombinant clone is exposed to conditions which induce a detectable signal, and detecting the detectable signal in the capillary tube to identify one or more capillaries containing the detectable signal thereby identifying the bioactivity or biomolecule of interest.
In another embodiment, the invention provides a method for identifying a bioactivity or biomolecule of interest by introducing a recombinant clone into a capillary tube of a capillary array, wherein each capillary tube of the capillary array has at least one wall defining a lumen for retaining the recombinant clone, and the at least one wall is made of a material having a low refractive index, and wherein the recombinant clone contains a substrate. The recombinant clone is exposed to conditions which causes the substrate to produce a detectable signal, and detecting the detectable signal in the capillary tube to identify one or more capillaries containing the detectable signal thereby identifying the bioactivity or biomolecule of interest.
In yet another embodiment, the invention provides a method for identifying a bioactivity or biomolecule of interest, by introducing a recombinant clone into a capillary tube of a capillary array, wherein each capillary tube of the capillary array has at least one wall defining a lumen for retaining the recombinant clone, and wherein the recombinant clone contains a substrate, exposing and the recombinant clone to conditions which causes the substrate to produce a detectable signal, and detecting the detectable signal in the capillary tube to identify one or more capillaries containing the detectable signals thereby identifying the bioactivity or biomolecule of interest.
In one embodiment, the invention provides a method for identifying a bioactivity or biomolecule of interest. The method includes introducing a substrate labeled with a detectable molecule and a recombinant clone into a capillary tube of a capillary array. Each capillary tube of the capillary array has at least one wall defining a lumen for retaining the substrate and the recombinant clone, and wherein the at least one wall is made of a material having a low refractive index. The method further includes culturing the capillary tube containing the substrate and the recombinant clone under conditions which allow interaction of the substrate and the recombinant clone to produce a detectable signal and detecting the detectable signal in the capillary tube to identify one or more capillaries containing the detectable signal thereby identifying the bioactivity or biomolecule of interest.
In another embodiment, the invention provides a method for identifying a bioactivity or biomolecule of interest by introducing a substrate labeled with a detectable molecule and a recombinant clone into a capillary tube of a capillary array, wherein each capillary tube of the capillary array has at least one wall defining a lumen for retaining the substrate and the recombinant clone and wherein each capillary tube in the array is separated from one another by an outer wall having a material with a low refractive index. The capillary tube containing the substrate and the recombinant clone are cultured under conditions which allow interaction of the substrate and the recombinant clone to produce a detectable signal and detecting the detectable signal in the capillary tube to identify one or more capillary tubes containing the detectable signal thereby identifying the bioactivity or biomolecule of interest.
In yet another embodiment, the invention provides a method for identifying a bioactivity or biomolecule of interest by introducing a substrate labeled with a detectable molecule and a recombinant clone into a capillary tube of a capillary array, wherein each capillary tube of the capillary array has at least a first wall and a second wall wherein the first wall defines a lumen for retaining the substrate and the recombinant clone and is made of a material having a high refractive index and the second wall surrounds the first wall and is made of a material having a low refractive index, wherein the second wall is in contact with at least one other capillary tube second wall. The capillary tube containing the substrate and the recombinant clone are cultured under conditions which allow interaction of the substrate and the recombinant clone to produce a detectable signal and detecting the detectable signal in the capillary tube to identify one or more capillary tubes containing the detectable signal thereby identifying the bioactivity or biomolecule of interest.
In another embodiment, the invention provides an automated capillary array system. The system includes a plurality of capillaries defining a capillary array, wherein each of the plurality of capillaries is separated from each other capillary in the array by at least one material having a low refractive index and wherein each capillary has openings at each end of the capillary. The system further includes at least one magnetic field apparatus in magnetic communication with the capillary array to cause movement of paramagnetic beads, an optical array in optical communication with at least one end of the capillary array that detects an optical signal produced from a sample in at least one capillary of the capillary array and a computer system in communication with the magnetic field apparatus and the optical array, wherein the computer system controls the magnetic field surrounding the capillary array and processes data detected by the optical array.
In another embodiment, the invention provides a method for identifying a compound of interest, by introducing a sample containing a plurality of compounds into a capillary tube of a capillary array, wherein each capillary tube of the capillary array has at least one wall defining a lumen for retaining the sample, and the at least one wall is made of a material having a low refractive index, and wherein the recombinant clone contains a substrate, exposing and the sample in the capillary tube to conditions which causes the compound of interest to produce a detectable signal, and detecting the detectable signal in the capillary tube to identify one or more capillaries containing the detectable signal thereby identifying the compound of interest.